Letrozole

Starting from the early follicular phase, commonly Days 2–4 of spontaneous menses.
or progesterone-induced withdrawal bleeding, the patients were treated with a dose of 5 mg letrozole (Jiangsu Hengrui Medicine Co., China) daily for 5 days. To assure the data comparability, we included only the patients treated with 5 mg letrozole per day for 5 days. Seven days after the first day that patients were administered letrozole, ultrasound monitoring and serum hormone analysis were performed. If there was no dominant follicle or the leading follicle was <14 mm, 75 IU human menopausal gonadotropin (HMG, Anhui Fengyuan Pharmaceutical Co.) every other day was prescribed. Thereafter, the endometrium, follicular development and sex hormone levels were monitored as necessary. If needed, the administered dose of HMG was increased by 37.5 IU incrementally, while the frequency of HMG use was increased up to daily. Moreover, a low dose of oral estrogen was added to improve the endometrium (EM) as needed. When at least one follicle reached a mean diameter of 17–18 mm, the EM thickness reached 7 mm, and ideally, when E2 levels were > 150 pg./ml, ovulation was triggered with human chorionic gonadotropin (hCG; 5,000 IU; Lizhu Pharmaceutical Trading Co.). IUI was then performed at the appropriate time.

Five of the 20 rats were randomly selected as the normal control group (oral administration of 1 mL normal saline for 28 days), and the remaining 15 rats were used to induce the PCOS model by giving 1 mg/kg Letrozole for 21 consecutive days [16 (link)]. After successful PCOS induction, 15 PCOS rats were randomly and equally divided into three groups: the PCOS group (oral administration of 1 mL saline for 28 days), the MET group (oral administration of 1 mL saline dissolved with 500 mg/kg metformin for 28 days) [16 (link)] and the SI group (oral administration of 1 mL saline dissolved with 100 mg/kg soy isoflavones for 28 days) [14 (link)]. The body weight of rats in each group was measured weekly for the duration of the study. After the 7^th week (Day 49), the rats were sacrificed to measure the weight and the ovary of each rat was removed for weighing and measuring long and short diameters. And then the ovarian volume [mm³ = (π/6)*long (mm)*short (mm)²] was calculated as described by Wang [17 (link)]. Additionally, samples of ovarian and vaginal tissues and serum were collected for subsequent index detection. Letrozole was purchased from Jiangsu Hengrui Medicine Co., Ltd.; SI were purchased from Xi’an Tianfeng Biotechnology Co., Ltd.; and metformin was provided by Bristol-Myers Squibb Company.

Pelvic ultrasound and serum hormone levels, including FSH, luteinizing hormone (LH), and estradiol (E₂), were assessed on the 2nd and 4th day of the menstrual cycle. Once the patients were confirmed to be in the early follicular phase, the administration of letrozole (Jiangsu Hengrui Medicine Co., China) at a dosage of 2.5–5 mg commenced on any of cycle days 3, 4, or 5 and continued for 5 days. A baseline ultrasound was conducted on the day of letrozole initiation, followed by subsequent ultrasounds 6–11 days later and then at intervals of 1–3 days as necessary until at least one follicle reached a mean diameter of ≥ 16 mm. In conjunction with ultrasound, the serum levels of E₂, LH, and/or progesterone were assessed as necessary. Upon identification of a mature follicle, ovulation was induced using human chorionic gonadotropin (hCG; Lizhu Pharmaceutical Trading Co., China), and intrauterine insemination (IUI) was scheduled to take place within 24–36 h following hCG administration. In the event of an LH surge (defined as an LH level exceeding 2.5 times the baseline value) [13 ], IUI was scheduled for the subsequent day. Even though we didn't use hCG, we called it a "trigger day."